-28th ISPSR POSTER

28th
International Symposium on
Pediatric Surgical Research
ABSTRACT BOOK
DUBLIN, IRELAND – 24-26 SEPTEMBER, 2015

28th International Symposium on Pediatric Surgical Research
1
FRIDAY, 25TH SEPTEMBER, 2015
SCIENTIFIC SESSION I
(PRIZE SESSION)

2
THREE- AND FOUR-DIMENSIONAL ANALYSIS OF ALTERED BEHAVIOR OF ENTERIC NEURAL CREST
DERIVED CELLS IN THE HIRSCHSPRUNG’S DISEASE MOUSE MODEL
N Nakazawa-Tanaka1,2
, K Miyahara2
, N Fujiwara2
, M Urao1
, C Akazawa3
, G J. Lane2
, A Yamataka2
1
Department of Pediatric Surgery, Juntendo Nerima Hospital
2
Department of Pediatric Surgery, Juntendo University School of Medicine, 3
Department of
Biochemistry and Biophysics, Graduate School of Health Care Science, Tokyo Medical and Dental
University
Tokyo, Japan
Email address for correspondence: nana.nakazawa@gmail.com
PURPOSE
The behavior of enteric neural crest-derived cells (ENCC) during enteric nervous system (ENS)
development is being gradually understood by the introduction of live-cell imaging technique.
However, many of the analysis to date are two- dimensional and the precise multidirectional
migration of ENCC has been challenging to interpret. Mice lacking the endothelin-B receptor gene,
EDNRB(-/-) mice, are widely used as a model for Hirschsprung’s disease (HD). We have recently
developed a SOX10 transgenic mouse to visualize ENCC with enhanced green fluorescent protein
(VENUS). By breeding these two models, we have created a VENUS-positive, SOX10 transgenic
mouse with a deletion of the EDNRB gene, SOX10-VENUS(+)/EDNRB(-/-) mouse, to investigate the
ENS in HD. The aim of this study was to investigate the behavior of migrating ENCC in the hindgut of
the SOX10-VENUS(+)/EDNRB(-/-) mouse by using three-dimensional and 4-dimensional (3D/4D)
image analysis software.
METHODS
To compare the ENCC behavior when the wavefront of ENCC reaches the mid-hindgut between HD
mouse and control, we harvested the fetal hindguts from SOX10-VENUS(+)/EDNRB(-/-) mice on
D15.5 and SOX10-VENUS(+)/EDNRB(+/+) mice on D12.5, which was used as control. Dissected
hindguts were cultured for 360 minutes and the time-lapse images were obtained using a confocal
laser-scanning microscope. Each ENCC at the wavefront was tracked after adjusting the longitudinal
axis of the gut to the Y axis and analyzed by using Imaris software (Bitplane, Zurich, Switzerland).

3
RESULTS
Track displacement (TD) of ENCC advancement in the rostral-caudal (vertical) plane was represented
by the Y axis (TD-Y). Horizontal ENCC advancement and depth of ENCC advancement were
represented by the X and Z axes as TD-X and TD-Z (Figure). Mean TD-Y was 34.56 µm in HD, but
63.48 µm in controls. Mean TD-X and TD- Z were similar in both groups. The mean track speeds were
decreased in HD (72.87 µm/h) compared to controls (248.29 µm/h).
CONCLUSIONS
Our results showed that the track speed and ENCC advancement in a rostral-caudal direction were
markedly decreased in the HD mice compared to controls. This technique provides added
information by tracking ENCC with depth perception, which has potential for further elucidating the
altered behavior of ENCC in HD.
Figure
Control
Control

4
CHARACTERIZATION OF VASCULOGENIC POTENTIAL OF HUMAN ADIPOSE-DERIVED ENDOTHELIAL
CELLS IN A THREE-DIMENSIONAL VASCULARIZED SKIN SUBSTITUTE
A. S. Klar1,2
, S. Güven3
, J. Zimoch1,2
, T. Biedermann1,2
, S. Böttcher-Haberzeth1,2,4
, C. Meuli-Simmen5
,
Ivan Martin3
, A. Scherberich3
, E. Reichmann1,2
, M. Meuli2,4
(1) Tissue Biology Research Unit, Department of Surgery, University Children’s Hospital Zurich,
Zurich, Switzerland
(2) Children’s Research Center, University Children’s Hospital Zurich, Zurich, Switzerland
(3) Department of Biomedicine, University Hospital of Basel, University of Basel, Basel, Switzerland
(4) Department of Surgery, University Children’s Hospital Zurich, Zurich, Switzerland
(5) Department of Plastic, Reconstructive, Esthetical and Hand Surgery, Kantonsspital Aarau, Aarau,
Switzerland
Corresponding author: Martin.Meuli@kispi.uzh.ch
Purpose: The clinical need for vascularized clinically applicable skin substitutes continues to grow. In
this study, we explored the use of human endothelial cells derived from freshly isolated adipose
stromal vascular fraction (SVF) in a 3D co-culture model of vascularized bio-engineered skin
substitute.
Methods: The SVF was isolated from human adipose tissue samples and keratinocytes from human
skin biopsies. The SVF, in particular endothelial cells were characterized using flow cytometry and
immuofluorescence analysis. Endothelial and mesenchymal progenitors from the SVF formed blood
capillaries after seeding into a 3D collagen type I hydrogel in vitro. Subsequently, human
keratinocytes were seeded on the top of those hydrogels to develop a vascularized dermo-epidermal
skin substitute.
Results: Flow cytometric analysis of surface markers of the freshly isolated SVF showed the
expression of mesenchymal/stromal cell-associated markers (CD44, CD73, CD90, CD105), stem cell
markers (CD133, CD49f), endothelial markers (CD31, CD34), and additionally hematopoietic markers
(CD14, CD45). Further analysis of adipose-derived endothelial cells (ECs) revealed the co-expression
of CD31, CD34, CD90, CD105, and partially CD146 on these cells. ECs were separated from adipose-
stromal cells (ASCs) using FACS sorting. ASCs and ECs cultured separately in a 3D hydrogel for 3
weeks did not form any vascular structures. Only if co-cultured, both cell types aligned to develop a
ramified vascular network in vitro with continuous endothelial lumen formation. Transplantation of
those 3D-hydrogels onto immuno-incompetent rats displayed a rapid connection of human
capillaries with the host vessels and formation of functional, blood-perfused mosaic human-rat
vessels within only 3-4 days.
Conclusions: Adipose tissue represents an attractive cell source due to the ease of isolation and
abundance of endothelial and mesenchymal cell lineages. Adipose-derived SVF cells exhibit the
ability to form microvascular structures in vitro and support the accelerated blood perfusion in skin
substitutes when transplanted.
Keywords: Adipose stem cells - Vascular network formation - Blood capillaries - Angiogenesis -
Stromal vascular fraction - Mesenchymal cells - Endothelial cells - Skin tissue engineering

5
Surfactant protein D attenuates lipopolysaccharide-induced inflammation in TLR-4-overexpressed
human intestinal cells
R Saka1) 2) 3)
, T Wakimoto3) 4)
, F Nishiumi3)
, T Sasaki1)
, S Nose1)
, M Fukuzawa5)
, T Oue1)
, I Yanagihara3)
, H
Okuyama2)
1) Department of Pediatric Surgery, Hyogo College of Medicine
2) Department of Pediatric Surgery, Osaka University Graduate School of Medicine
3) Department of Developmental Medicine, Osaka Medical Center and Research Institute for
Maternal and Child Health
4) Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine
5) Department of Pediatric Surgery, Osaka Medical Center and Research Institute for Maternal and
Child Health
Address correspondence;
Hiroomi Okuyama, MD, Ph. D
Department of Pediatric Surgery, Osaka University Graduate School of Medicine
Address: 2-2 Yamadaoka, Suita-shi, Osaka, Japan 5650871
E-mail: okuyama@pedsurg.med.osaka-u.ac.jp
Abstract
PURPOSE: Necrotizing enterocolitis (NEC) is a devastating disease specific to preterm infants.
Recently, it has been reported that overexpressed toll-like receptor (TLR)-4 in the immature intestine
is associated with the development of NEC. Surfactant protein (SP)-D is a member of the collectin
family and plays an important role in innate immunity, particularly in the airways. Although SP-D also
exists in the intestines, little is known about its function. The aim of this study was to investigate
whether SP-D attenuates inflammation in TLR4 overexpressed intestinal cells.
METHODS: All experimental procedures were performed using the human intestinal cell line INT407
originally derived from human embryonal intestines. Platelet activating factor (PAF), reported to be
elevated in NEC patients, was used to overexpress TLR-4 in this cell line to mimic an immature
intestine. Following stimulation with PAF, the TLR4 expression was measured using quantitative real-
time PCR (normalized to GAPDH). The degree of inflammation induced by PAF and
lipopolysaccharide (LPS) and the effects of SP-D were assessed with interleukin 8 (IL-8) measured by
enzyme-linked immunosorbent assay (ELISA). The results of the quantitative RT-PCR and ELISA
analyses are expressed as the mean ± SD.
RESULTS: Following treatment with 5 µM of PAF, the expression levels of TLR4 mRNA were
significantly upregulated (3.69+/-0.28) compared with that observed in the cells treated without PAF
stimulation (1.00+/-0.10) (p<0.001). Stimulation with 5 µM of PAF and 100ng/ml of LPS resulted in
significantly higher IL-8 production (1959.3+/-52.3 pg/ml) compared with control (141.2+/-12.4
pg/ml), LPS alone (167.3+/-65.8 pg/ml) and PAF alone (1527.2+/-129.4 pg/ml) (P<0.05) The IL-8
expression with the stimulation of PAF and LPS (1590.1+/-319.3 pg/ml) was attenuated by
pretreatment with 20µg/ml of SP-D (1161.6+/-131.6 pg/ml) (p<0.05).

6
CONCLUSION: SP-D attenuates LPS-induced IL-8 production in TLR4 overexpressed intestinal cells.
These findings suggest that SP-D may have a protective effect in the development of NEC in preterm
infants.

7
NOTOCHORD MANIPULATION DOES NOT IMPACT OESOPHAGEAL AND TRACHEAL FORMATION
FROM ISOLATED FOREGUT IN 3D EXPLANT CULTURE.
Danielle Mc Laughlin1, 2
, Paula Murphy1
, Prem Puri2
(1)
School of Natural Sciences, Trinity College Dublin,
(2)
National Children’s Research Centre, Our Lady’s Children’s Hospital Crumlin,
DUBLIN, IRELAND,
Background: Tracheo-oesophageal malformations result from disturbed foregut separation during
early development. The notochord, a specialised embryonic structure, forms immediately adjacent
to the dividing foregut. In the Adriamycin mouse model of oesophageal atresia, foregut and
notochord abnormalities co-exist, and the site and severity of foregut malformations closely
correlate to the position and extent of the notochord defects. Notochord and foregut abnormalities
also co-exist in the Noggin Knockout mouse as well in a small number of human cases. The
notochord is a source of powerful molecular signals during early embryogenesis, being particularly
important for neural crest development. The influence of the notochord signaling on the adjacent
foregut is not known. It was the purpose of this study to examine the impact of notochord
manipulation on foregut separation using a robust 3D explant method for culturing isolated foregut
which permits oeosphageal and tracheal formation in vitro.
Methods: Foregut was micro-dissected from embryonic day 9 mice (License B100/4447 Irish
Medicines Board), embedded in collagen and cultured for 48 hours with native notochord intact
(n=6), notochord removed (n=10) or additional notochord transplanted from stage matched controls
(n=8). Specimens were analysed for foregut morphology and molecular patterning using
immunohistochemistry for Hnf3b (an endoderm marker) and Sox2 (a notochord and oesophageal
marker) on cryosections.
Results: Foregut separation into distinct oesophagus and trachea was observed in isolated foregut
specimens with or without their native notochord (Fig A,B). In specimens with additional notochord
transplants foregut morphology and molecular patterning was comparable to controls whether or

8
not the native notochord was maintained (Fig C to G). In particular foregut separation was not
disrupted by the transplantation of additional notochord at the dorsal foregut endoderm.
Conclusion: The relationship between the embryonic foregut and notochord is complex and ill-
defined however the notochord does not contribute essentially to oesophagus and trachea
formation beyond E9 in the mouse, and the transplantation of additional notochord does not disrupt
foregut separation in 3D explant culture.
Figure: Immunohistochemistry for Hnf3b and Sox2 on isolated foregut explant cryosections with (A)
or without their native notochord (B) showing foregut (fg) division into oesophagus (oe) and trachea
(tr). Transplant of additional notochord (D,E,G) does not impact foregut separation or molecular
patterning compared to controls (C and F).

9
IMPAIRED CYTOSKELETAL ARRANGEMENTS AND FAILURE OF VENTRAL BODY WALL CLOSURE IN
CHICK EMBRYOS TREATED WITH ROCK INHIBITOR (Y-27632)
Johannes W DUESS1,2
, Prem PURI1,2,3
, Jennifer THOMPSON1,2
(1)
National Children’s Research Centre, Our Lady’s Children’s Hospital, Dublin, Ireland
(2)
School of Medicine and Medical Science, University College Dublin, Ireland
(3)
Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Ireland
Purpose: Rho-associated kinase (ROCK) signaling regulates numerous fundamental developmental
processes during embryogenesis, primarily by controlling actin-cytoskeleton assembly and cell
contractility. ROCK knockout mice exhibit a ventral body wall defect (VBWD) phenotype due to
disorganization of actin filaments at the umbilical ring. However, the exact molecular mechanisms
leading to VBWD still remain unclear. Improper somitogenesis has been hypothesized to contribute
to failure of VBW closure. Adhesion molecules and connexins are involved in multiple morphological
events, including somite formation, through cytoskeletal anchorage. Vinculin and microtubules are
crucial for normal cell migration by binding to adhesion molecules and connexins. We designed this
study to investigate the hypothesis that administration of ROCK inhibitor (Y-27632) disrupts
cytoskeletal arrangements in morphology during early chick embryogenesis, which may contribute
to the development of VBWD.
Methods: At 60h incubation, chick embryos were explanted into shell-less culture and treated with
50µL of vehicle for controls (n=33) or 50µL of 500µM of Y-27632 for the experimental group (Y-27,
n=56). At 8h post-treatment, RT-PCR was performed to evaluate mRNA levels of N-cadherin, E-
cadherin and connexin43. Immunofluorescence confocal microscopy was performed to analyze the
expression and distribution of actin, vinculin and microtubules in the neural tube and somites. A
further cohort of embryos was treated in ovo by dropping 50µL of vehicle or 50µL of 1000µM of Y-
27632 onto the embryo and allowing development to 14d for further assessment.
Results: Gene expression levels of N-cadherin, E-cadherin and connexin43 were significantly
decreased in treated embryos compared with controls (p<0.05) (Fig.1). Thickened actin filament
bundles were recorded in the neural tube of Y-27 embryos. In somites, cells were disorientated with
reduced actin distribution in affected embryos (Fig.2). Vinculin was over-expressed and clumped in
the neural tube and somites (Fig.3), whereas reduced expression of tubulin was observed in Y-27
embryos compared with controls (Fig.4). At 14d of development, affected embryos presented with
an enlarged umbilical ring and herniation of abdominal contents, associated with malformed lower
limbs (Fig.5).

10
Conclusion: ROCK inhibition impairs cytoskeletal arrangement during early chick embryogenesis,
which may contribute to failure of anterior body wall closure causing VBWD at later stages of
development.

11
INTESTINAL MICROBIOTA OF INFANTS REQUIRING SURGERY ANALYSIS USING NEXT-GENERATION
DNA SEQUENCING
H. Murakami1
, Y. Shimomura2
, M. Matsumoto2
, GJ.Lane1
, A. Yamataka1
, M. Okawada1
1) Department of Pediatric General and Urogenital Surgery
Juntendo University School of Medicine, Tokyo, Japan
2) Research Laboratories, Kyodo Milk Industry Co., Ltd, Tokyo, Japan
E-mail address of corresponding author: manabu-o@juntendo.ac.jp
PURPOSE: Development of the intestinal environment in infants is characterized by rapid, large
changes in the abundance, diversity, and composition of microbiota under the influence of various
factors that strongly influence both adult intestinal microbiota and development of the adult
intestinal immune system. Probiotic therapy has been proposed as an effective way to enhance the
intestinal environment during times of stress but there are few reports about probiotic use in
children. The aim of this study was to conduct a comprehensive investigation of the intestinal
microbiota of infants undergoing surgery using a next-generation DNA sequencing system and assess
the value of probiotic administration.
METHODS: We studied 8 infants requiring surgery. All were born at our institution. The probiotic
preparation used was Bifidobacterium lactis LKM512 (LKM) 1.0 g/day administered orally. The 8
subjects were divided into two groups according to LKM use; LKM+ (n=4) and LKM- (n=4). As controls
we included 2 infants who did not require surgery and who were not given LKM (C; n=2). Clinical
examinations were performed according to our standard postoperative protocol and stool
specimens (0.5g) were collected 5 times (after birth, and on days 3, 7, 10, and 14 after surgery).
Extracted fecal DNA underwent PCR amplification for 16S rRNA amplicon library preparation and all
stool specimens were sequenced with an Ion TorrentTM
PGMTM
Sequencer.
RESULTS: Clinical status and all laboratory data were similar for LKM+ and LKM-. All stool specimens
were successfully sequenced using only 20mg of stool. The microbiota in C stool was relatively
varied, with Enterobacteriaceae, Bacteroidaceae, Bifidobacteriaceae, and Streptococcaceae
identified in descending order of abundance. There were significantly more Enterococcaceae,
Streptococcaceae, and Staphylococcaceae in both LKM+ and LKM- compared with controls.
Unexpectedly, there were significantly more Bifidobacteriaceae in LKM- (39.8%) than LKM+ (12.8%)
(p<0.005; Figure).
CONCLUSION: The impact of surgical stress on intestinal microbiota in infants would appear to be
considerable and warrants further investigation and the role of probiotic administration in infants
requires clarification. New-generation DNA sequencing was particularly effective for identifying stool
microbiota and will facilitate larger scale studies by reducing costs and labor.

12

13
Intraoperative acidosis and hypercapnia during thoracoscopic repair of esophageal
atresia/tracheoesophageal fistula
Augusto Zani, Ruben Lamas-Pinheiro, Irene Paraboschi, Giovanni Cobellis, Justyna Wolinska, Elke
Zani-Ruttenstock, Agostino Pierro
Division of General and Thoracic Surgery, The Hospital for Sick Children, Toronto, Ontario, Canada
PURPOSE: It has been reported that thoracoscopic repair of esophageal atresia/tracheoesophageal
fistula (EA/TEF) is associated with severe intraoperative hypercapnia and acidosis. The aim of the
present study was to confirm this phenomenon in a larger population from a North-American
Institution.
METHODS: Following ethical approval (REB1000046653), we retrospectively reviewed the medical
charts of all neonates who underwent open or thoracoscopic EA/TEF repair between 2004 and 2014
at our Institution. Only patients with available intraoperative arterial gas values were included in this
analysis. Data were compared using paired/unpaired t-tests and linear regression analysis and are
reported as mean ± SD or median (range).
RESULTS: During the study period, 205 infants underwent open (n=180) or thoracoscopic (n=25)
EA/TEF repair. Intraoperative arterial gas values were recorded in 62 (34%) open and in 14 (56%)
thoracoscopic operations. There were no differences in birth weight (p=0.2) or gestational age
(p=0.06). Both groups had similar preoperative pH (p=0.3, Figure) and PaCO2 (p=0.2) but developed
intraoperative acidosis (open: preop vs. intraop p<0.0003; thoracoscopic: preop vs. intraop
p<0.0001) and hypercapnia (open: preop vs. intraop p=0.008; thoracoscopic: preop vs. intraop
p=0.03). Intraoperatively, infants undergoing thoracoscopic repair had lower pH than those having
open surgery (p= 0.01, Figure) and higher levels of hypercapnia (p=0.03). In none of the 5 (20%)
patients who had a conversion to open surgery, this was due to ventilatory concerns. Postoperative
ventilation was continued for 2 days (0-21) after open surgery and for 3 days (1-8) after
thoracoscopy (p=0.9). There was no correlation between intraoperative acidosis and postoperative
ventilation duration (r2
= 0.05).
CONCLUSIONS: Neonates undergoing surgical repair of EA/TEF develop intraoperative acidosis and
hypercapnia regardless the approach used. However, this phenomenon is more severe during
thoracoscopic repair. The effects of acidosis and hypercapnia on brain development are unknown.
Novel modalities to reduce intraoperative gas derangements particularly during thoracoscopic
surgery need to be established.

14
REDUCED EXPRESSION OF VOLTAGE-GATED KV11.1 (HERG) K+ CHANNELS IN AGANGLIONIC COLON
IN HIRSCHSPRUNG’S DISEASE
Tomuschat C, O’Donnell AM, Coyle D, Puri P
National Childrens Research Centre, Ireland, Dublin
Our Lady’s Children’s Hospital, Crumlin, Dublin
prem.puri@ncrc.ie
Purpose: The pathophysiology of Hirschsprung’s Disease (HD) is not fully understood. There is no
clear explanation for the occurrence of the spastic or tonically contracted aganglionic segment of
bowel. Kv11.1 (HERG) channels play a key role in the regulation of the resting membrane potential
as well as affecting either the force or frequency of contraction of smooth muscles. We designed this
study to investigate the expression and characterization of HERG channels in the normal colon and in
the colon of patients with HD.
Methods: We investigated Kv11.1 protein expression in both the ganglionic and aganglionic regions
of HD patients (n=10) versus normal control colon (n=10). The distribution was assessed by using
immunofluorescence and confocal microscopy. Gene and protein expression was quantified using
quantitative real-time polymerase chain reaction, western blot analysis and densitometry.
Results: Confocal microscopy of the normal colon revealed strong Kv11.1 channel expression in the
enteric neurons, interstitial cells of Cajal (ICCs) and PDGFRα+
cells (Figure 2). HERG expression was
markedly decreased in the aganglionic bowel. Colonic HERG gene expression levels were significantly
decreased in HD specimen compared to controls (p < 0.003). Western blotting revealed decreased
colonic HERG protein expression in aganglionic bowel compared to controls (Figure 1).
Conclusions: We demonstrate for the first time the expression and distribution of HERG channels in
enteric neurons, ICCs and PDGFRα+
The decreased expression of Kv11.1 (HERG) in the aganglionic
colon may be responsible for the increased tone in the aganglionic narrow spastic segment of bowel.
Figure 1

15
Figure 2

16
ENHANCED LIVER GENE EXPRESSION OF BILIARY ABC STEROL TRANSPORTERS IS LINKED WITH
DEVELOPMENT OF GALLSTONES IN CHILDREN
A Koivusalo, A Mutanen, MP Pakarinen
Section of pediatric surgery, Pediatric Liver and Gut Research Group, Children´s Hospital, University
of Helsinki, Finland
mikko.pakarinen@hus.fi
PURPOSE: To elucidate the pathogenesis of increasingly common pediatric cholelithiasis, we
assessed liver RNA expression of canalicular bile transporters and their upstream nuclear receptor
regulators in relation to gallstone sterol content and surrogate serum markers of cholesterol
synthesis (precursors) and absorption (plant sterols).
METHODS: After ethical approval, twenty children with gallstones (11 females), median age 11 (IQR
8.2) years and six with predisposing conditions underwent cholecystectomy, intraoperative liver
biopsy and serum sampling. Liver RNA expression of biliary sterol (ABCG5, ABCG8), phospholipid
(ABCB4), bile acid (ABCB11) and bilirubin (ABCC2) transporters and their upstream regulators (LXR
and FXR) was measured with real time RT-PCR (ΔΔCt method). Serum and gallstone concentrations
of cholesterol, cholesterol precursors (cholestenol and lathosterol) and plant sterols (stigmasterol,
avenasterol, sitosterol, and campesterol) were measured using gas-liquid chromatography.
Gallstones were classified as cholesterol gallstones (CGS, cholesterol > 35% of weight) or pigment
stones (PS). Liver biopsies of six healthy donor livers (n = 6), and serum from 82 healthy children
served as controls.
RESULTS: As shown in the figure, RNA expression of canalicular sterol transporters (ABCG5 and
ABCG 8) and their upstream regulator LXR were 1.5– 2.7 times higher in gallstone patients than in
controls (p < 0.02– 0.0003), but were not related with stone or serum cholesterol content,
predisposing conditions, age, sex or body mass index. Expression of biliary phospholipid, bile acid
and bilirubin transporters were unaltered. Patients with CGS (n = 10) had 1.7 – 2.2 times higher
serum cholestenol and lathosterol (p < 0.0001) and 1.3 – 2.0 lower stigmasterol and sitosterol (p <
0.03-0.0001), than patients with PS (n=10) or controls (n=82). CGS contained 4 – 24 times more
cholestenol and lathosterol (ug /mg cholesterol) than PS (p = 0.0003 – 0.0005), whereas PS displayed
6.7 times higher concentration of stigmasterol (p = 0.0005).
CONCLUSION: Enhanced liver gene expression of LXR and canalicular sterol transport proteins
ABCG5 and ABCG8 suggest that increased biliary secretion of sterols, including cholesterol and plant
sterols, is characteristic to pediatric gallstone disease. CGS developed in patients with increased
cholesterol synthesis, whereas patients with normal cholesterol synthesis developed PS enriched by
plant sterols.
0
,5
1
1,5
2
2,5
3
3,5
Foldexpression(mean,95%CI)
LXR ABCG5 ABCB8
control
patient
*
*
*
* P < 0.04 vs controls

17
BLADDER EXSTROPHY - A NATIONWIDE REGISTER STUDY ON MATERNAL AND FETAL FACTORS IN
SWEDEN 1973-2011
G R Engberg1,2
, Ä Mantel3
, M Fossum1,2
, A Nordenskjöld1,2
1.
Department of Women’s and Children’s Health and Center of Molecular Medicine, Karolinska
Institutet, Stockholm, Sweden
2.
Department of Pediatric Surgery, Urology Section, Astrid Lindgren Children’s Hospital, Karolinska
University Hospital, Stockholm, Sweden
3.
Clinical Epidemiology Unit, Department of Medicine, Solna (MedS), K2, Karolinska Institutet,
Stockholm, Sweden
gisela.reinfeldt.engberg@ki.se
PURPOSE
Bladder exstrophy is a rare congenital malformation where the underlying cause is largely unknown,
but both environmental and genetic mechanisms are involved. The malformation often require
multiple interventions by pediatric surgical teams from the newborn period into adolescence and
sometimes adulthood.
The aim of this study was to conduct a nationwide study related to maternal and fetal risk factors of
bladder exstrophy in Sweden 1973-2011.
METHODS
120 patients were identified in the Medical Birth Register with ICD codes; 753.50, 753F and Q64.1.
Cases were matched with 5 controls per patient for calendar year of delivery and sex. A case-control
study was performed by linkage of national registers. We studied additional malformations and
environmental factors such as maternal age, BMI, smoking, parity, seasonal differences and assisted
conception. We also studied delivery mode, birth weight, gestational week at birth, Apgar, survival
rate and length of hospital stay.
RESULTS
The prevalence was 2.98:100 000 live births with a male-to-female ratio 1.14:1. Bladder exstrophy
was an isolated malformation in 93% of the cases excluding inguinal hernia and cryptorchidism. 41%
had received surgery for inguinal hernia, 63% of the male and 16% of the female cases. In addition,
11% of the boys had undergone surgery for non-descended testis but none of them were born
premature or SGA.
There was a tendency of high maternal age, extremes of BMI and smoking as risk factors for bladder
exstrophy. Parity, seasonal differences and assisted conception showed no tendency of being risk
factors. Delivery mode, birth weight, gestational age at birth, Apgar and survival rate corresponded
to controls but patients stayed a few days longer at the hospital after birth.
CONCLUSIONS
This nationwide study demonstrates a prevalence of 3:100 000 live births with an almost equal sex
ratio. Maternal high age, extremes of BMI and smoking appear to be associated with higher risk. The
majority of the cases were isolated without major associated malformations. A high percentage of
the affected children received surgery for inguinal hernia and non-descended testis. Bladder
exstrophy did neither affect birth weight, gestational age at birth, Apgar nor survival rate.

18
SCIENTIFIC SESSION II

19
A NOVEL TREATMENT FOR HYPOPLASTIC LUNG ASSOCIATED WITH CONGENITAL DIAPHRAGMATIC
HERNIA BY PRENATAL MATERNAL ADMINISTRATION OF SLOW-RELEASE SYNTHETIC PROSTACYCLIN
AGONIST IN EXPERIMENTAL RAT MODEL
S. Umeda1
, S. Miyagawa2
, S. Fukushima2
, A. Harada2
, A. Saito2
, Y. Sakai2
, Y. Sawa2
, H. Okuyama1
1
Department of Pediatric Surgery
2
Department of Cardiovascular Surgery
Osaka University Graduate School of Medicine
2-2 Yamadaoka, Suita, Osaka, Japan
E-mail address of corresponding author; sumeda@pedsurg.med.osaka-u.ac.jp
PURPOSE: The major pathogenesis of congenital diaphragmatic hernia (CDH) is delayed lung
development during fetal period, which leads to postnatal lethal lung hypoplasia and pulmonary
hypertension (PH). It was recently reported that administration of sildenafil or vascular-endothelial
growth factor (VEGF) enhanced development of fetal hypoplastic lungs in a rat model of CDH. On the
other hand, ONO-1301, a synthetic prostacyclin receptor agonist, has been reported to upregulate
proangiogenic and anti-inflammatory factors in a variety of pathologies including a rat model of PH.
We therefore hypothesized that prenatal maternal administration of slow release form of ONO-1301
(ONO-1301SR), which is polylactic-co-glycolic acid copolymer (PLGA)-polymerized ONO-1301, would
attenuates lung hypoplasia and pulmonary vascular remodeling associated with nitrofen-induced
CDH.
METHODS: Pregnant Sprague-Dawley rats were gavage fed with nitrofen to produce a CDH-relating
lung hypoplasia model on embryonic day (E) 9.5, while control rats received vehicle only. At the
same day, ONO-1301SR and placebo were injected subcutaneously. Rats were randomized into 3
groups: control (n=4), nitrofen treatment (n=7), nitrofen+ONO1301SR treatment (n=5). At term
(E21.5), the fetal lungs were harvested for further pathobiological evaluation. In nitrofen and
nitrofen+ONO1301SR group, only those which developed CDH fetuses were analyzed.
RESULTS: The incidence of nitrofen-induced CDH was not influenced by prenatal ONO-1301SR
administration. One-seventh of maternal plasma concentration of ONO-1301 was transferred to
fetal plasma at E21.5, suggesting this molecule has an efficient placental permeability. Prostacyclin
receptor expression was localized in pulmonary arterial smooth muscle cells in the fetus. Lung-to-
body weight ratio (%) in the CDH+ONO group (1.88±0.07) was greater than that in the CDH group
(1.60±0.04, p<0.01). Histologically, medial wall thickness in the CDH+ONO group was two-third
thinner than that in the CDH group (p<0.01). In addition, mean linear intercept, an indicator of
pulmonary airspace, were 1.5 times greater in the CDH+ONO group than that in the CDH group
(p<0.05). These changes were associated with a greater expression of VEGF in the CDH+ONO group.
CONCLUSION: Prenatal ONO-1301SR treatment attenuated lung hypoplasia and pulmonary vascular
remodeling in experimental nitrofen-induced CDH, indicating potential of this treatment for
pathologies having lung hypoplasia associated with CDH.

20
Antenatal retinoic acid administration promotes lung growth by augmenting retinol levels in the
nitrofen model of congenital diaphragmatic hernia
Balazs Kutasy1
, Florian Friedmacher1
, Lara Pes2
, David Coyle1
, Takashi Doi1
, Francesca Paradisi2
, Prem
Puri1
1:Our Lady's Children's Hospital, National Children's Research Centre, Dublin, Ireland, 2:Centre for
Synthesis & Chemical Biology, School of Chemistry & Chemical Biology, University College Dublin,
Dublin, Ireland
Background: Low pulmonary retinol levels and disrupted retinoid signaling pathway (RSP) has been
implicated in the pathogenesis of congenital diaphragmatic hernia (CDH) and associated pulmonary
hypoplasia (PH). It has been demonstrated that nitrofen disturbs the retinol transport. Several
studies have demonstrated that prenatal treatment with retinoic acid (RA) can reverse PH in the
nitrofen-induced CDH model. We hypothesized that maternal administration of RA can increase fetal
serum and pulmonary retinol levels and the gene expression of main components of the RSP in a
nitrofen model of CDH.
Methods: Pregnant rats were exposed to either olive oil or nitrofen on day9 of gestation (D9) and
sacrificed on D21. RA was given intraperitoneally on D18,D19 and D20. Fetal left lungs were
dissected on D21 and divided into four groups: control, control+RA, CDH and CDH+RA. Lungs and
body weight were measured. Retinol levels were measured using HPLC. Expression levels of the
primary RSP-genes (RBPI, RALDH3, RARa, RARb, RXRa) were determined using qRT-PCR and
immunohistochemistry.
Results: The incidence rate of diaphragmatic hernia was similar in both CDH and CDH+RA group (
77/131, 58.7% vs 73/127, 57.4%). Both body and lungs weight were significantly higher in the
CDH+RA group compared to CDH group (p<0.05)(Table). Serum and pulmonary retinol levels were
significantly increased in control+RA compared to control (p<0.05)(Table) and in CDH+RA compared
to CDH (p<0.05)(Table). The relative mRNA expression levels of RSP genes were significantly
increased in control+RA compared to control (p<0.05) and in CDH+RA compared to CDH (p<0.05)
(Table). Markedly increased immunoreactivity of RSP was observed in control+RA and CDH+RA lungs
compared to control and CDH (Figure).
Conclusions: Our data shows that prenatal treatment with RA elevates fetal retinol levels in lungs
and serum. Enhanced retinol levels accompanied by the increased gene expression of main
components of RSP suggest that retinol triggered RSP-activation may result in reversal of pulmonary
hypoplasia in the nitrofen model.

21
Weight (g) Control Control+RA CDH CDH+RA
Left lungs 0.054±0.002 0.059±0.003 0.025 ±0.001*,
** 0.039 ±0.01
Right lungs 0.078 ±0.004 0.085 ±0.001 0.051±0.002*,
** 0.069±0.002
Body 5.1 ±0.08 6.2 ±0.1* 4.68±0.1*,
** 4.98±0.07
*: vs control, p<0.05; **: vs CDH+RA, p<0.05
Retinol level Control Control+RA CDH CDH+RA
Serum 0.0872±0.001 0.181±0.001* 0.0151±0.003* 0.195±0.004*,
**
Left lungs 0.0816 ± 0.01 0.121±0.01* 0.0717 ± 0.01* 0.151±0.01*,
**
*: vs control, p<0.05; **: vs CDH+RA, p<0.05
RBP1 RALDH3 RARa RARb RXRa
Control (n=8) 0.78 ± 0.09** 0.03 ± 0.01** 0.54 ± 0.04** 0.08 ± 0.01** 0.14 ± 0.01**
Control+RA (n=8) 1.52 ± 0.07* 0.04 ± 0.01* 0.94 ± 0.04* 0.13 ± 0.01* 0.38 ± 0.01*
CDH (n=8) 1.02 ± 0.40 0.07 ± 0.01 0.68 ± 0.03 0.09 ± 0.01 0.36 ± 0.03
CDH+RA (n=8) 1.71 ± 0.10** 0.10 ± 0.01** 1.22 ± 0.08** 1.99 ± 0.01** 0.48 ± 0.03**
*: vs control, p<0.05; **: vs CDH, p<0.05;

22

23
MESENCHYMAL EXPRESSION OF THE FRAS1/FREM2 GENE UNIT IS DECREASED IN THE DEVELOPING
DIAPHRAGM OF NITROFEN-INDUCED CONGENITAL DIAPHRAGMATIC HERNIA
T. Takahashi1
, F. Friedmacher1
, J. Zimmer1
, P. Puri1
1
National Children’s Research Centre, Our Lady’s Children’s Hospital, Crumlin, Dublin, Ireland
E-mail: prem.puri@ncrc.ie
PURPOSE: Developmental mutations that inhibit normal formation of extracellular matrix (ECM) in
fetal diaphragms have been identified in congenital diaphragmatic hernia (CDH). FRAS1 and FRAS1-
related extracellular matrix2 (FREM2), which encode important ECM proteins, are secreted by
mesenchymal cells during diaphragmatic development. The FRAS1/FREM2 gene unit has been
shown to form a ternary complex with FREM1, which plays a crucial role during formation of human
and rodent diaphragms. Furthermore, it has been demonstrated that the diaphragmatic expression
of FREM1 is decreased in the nitrofen-induced CDH model. We hypothesized that FRAS1 and FREM2
expression is decreased in developing diaphragms of fetal rats with nitrofen-induced CDH.
METHODS: Timed-pregnant rats were exposed to either nitrofen or vehicle on gestational day 9
(D9), and fetuses were harvested on selected time-points D13, D15 and D18. Dissected diaphragms
(n=72) were divided into two groups: control and nitrofen-exposed samples (n=12 per time-point
and experimental group, respectively). Diaphragmatic gene expression levels of FRAS1 and FREM2
were analyzed by qPCR. Immunofluorescence-double-staining for FRAS1 and FREM2 was combined
with the mesenchymal marker GATA4 in order to evaluate protein expression and localization in
developing fetal diaphragms.
RESULTS: Relative mRNA expression of FRAS1 and FREM2 were significantly reduced in
pleuroperitoneal folds of nitrofen-exposed fetuses on D13 (1.76±0.86 vs. 3.09±1.15; p<0.05 and
0.47±0.26 vs. 0.82±0.36; p<0.05), developing diaphragms of nitrofen-exposed fetuses on D15
(1.45±0.80 vs. 2.63±0.84; p<0.05 and 0.41±0.16 vs. 1.02±0.49; p<0.05) and fully muscularized
diaphragms of nitrofen-exposed fetuses on D18 (1.35±0.75 vs. 2.32±0.92; p<0.05 and 0.37±0.24 vs.
0.70±0.32; p<0.05) compared to controls. Confocal laser scanning microscopy revealed markedly
diminished diaphragmatic FRAS1 and FREM2 immunofluorescence, which was associated with
reduced proliferation of diaphragmatic mesenchymal cells in nitrofen-exposed fetuses on D13, D15
and D18 compared to controls (Figure).

24
CONCLUSION: Decreased mesenchymal expression of FRAS1 and FREM2 in the nitrofen-induced
CDH model may cause failure of the FRAS1/FREM2 gene unit to activate FREM1 signaling, disturbing
the formation of diaphragmatic ECM and thus contributing to the development of diaphragmatic
defects in CDH.

25
UPREGULATION OF S1P1 RECEPTOR IN THE PULMONARY VASCULATURE OF NITROFEN-INDUCED
CONGENITAL DIAPHRAGMATIC HERNIA
J. Zimmer1
, T. Takahashi1
, J. W. Duess1,2
, A.D. Hofmann1,3
, P. Puri1,2
1
National Children's Research Centre, Our Lady's Children's Hospital, Dublin,Ireland
2
School of Medicine and Medical Science and Conway Institute of Biomolecular and Biomedical
Research, University College Dublin, Ireland
3
Department of Pediatric Surgery, Hannover Medical School, Hannover, Germany
Purpose: The high morbidity and mortality in neonates with congenital diaphragmatic hernia (CDH)
is attributed to severe pulmonary hypoplasia and persistent pulmonary hypertension (PH).
Sphingolipids have been demonstrated to play a crucial role in pulmonary development. The
sphingosine kinase 1 (SphK1) is an oncogenic enzyme, which modulates the synthesis of sphingolipid
sphingosine-1-phosphate (S1P). S1P is a key mediator regulating cell proliferation, migration and
angiogenesis via different receptor subtypes, S1P1, S1P2 and S1P3. Recently, the SphK1/S1P
pathway has been shown to be involved in adult human pulmonary arterial hypertension by
promoting pulmonary artery smooth muscle cell (SMC) proliferation. The aim of this study was to
investigate which S1P receptors are responsible for S1P induced abnormal pulmonary vascular
remodelling in the nitrofen-induced CDH model.
Methods: Pregnant Sprague Dawley rats were exposed to nitrofen or vehicle on D9 of gestation.
Fetuses were sacrificed on D21 and divided into nitrofen and control group (n = 12). Pulmonary RNA
was extracted and mRNA levels of SphK1, S1P1, S1P2 and S1P3 were determined by quantitative
real-time PCR. Western blotting and confocal-immunofluorescence microscopy were performed to
determine pulmonary protein expression as well as vascular localization of expressed SphK1, S1P1,
S1P2 and S1P3.
Results: Confocal-microscopy revealed an increased pulmonary vascular expression of S1P1 (Fig.1)
and a decreased expression of S1P2 and S1P3 in lungs of nitrofen-exposed fetuses compared to
controls. These results were confirmed by western blotting (Fig.2) and quantitative real-time PCR.
SpHK1 expression was not found to be altered in treated rats compared to controls.
Conclusion: We show for the first time the increased S1P1 receptor pulmonary vascular expression
in the nitrofen induced CDH. These results suggest that S1P1 receptor is involved in the S1P induced
pulmonary vascular remodeling resulting in PH in this model.

26
Fig.1

27
POSSIBLE ROLE OF INCREASED OXIDATIVE STRESS IN PULMONARY HYPERTENSION IN
EXPERIMENTAL DIAPHRAGMATIC HERNIA
R Aras-López1
, JA Tovar1,2
, L Martínez1,2
1
INGEMM and Idipaz Research Laboratory, Hospital Universitario La Paz, Madrid, Spain.
2
Department of Pediatric Surgery, Hospital Universitario La Paz, Madrid, Spain.
rosa.aras@hotmail.com
PURPOSE
Congenital diaphragmatic hernia (CDH) is one of the causes of respiratory failure in newborns due to
lung hypoplasia and pulmonary abnormalities leading to pulmonary hypertension (PH). NAD(P)H
oxidase (Nox), is a family of isoenzymes, composed of seven members Nox1-5, and the dual oxidases
(Duox) Duox1 and Duox2, that generate reactive oxygen species (ROS) such as anion superoxide
(•O2
−
) and hydrogen peroxide (H2O2) in pulmonary vascular cells which can contribute to PH-induced
vascular dysfunction. Our aim is to examine whether PH-associated to CDH is due to a dysregulation
of ROS production in lungs from CDH fetuses.
METHODS
Pregnant rats received either 100 mg nitrofen or vehicle on E9.5. Fetuses were sacrificed on E21 and
divided into control and CDH groups. Fetal lungs were recovered to determine (1) Nox activity by a
lucigenin-enhanced chemiluminescence assay, (2) H2O2 production by Amplex Red assay and (3)
mRNA levels of Nox1, Nox2 and Nox4 isoenzymes by real-time polymerase chain reaction (RT-PCR).
RESULTS
Nox activity and Nox1 and Nox2 mRNA levels were increased in the lungs of fetuses with CDH.
However, there were no changes in H2O2 production and Nox4 mRNA levels.
CONCLUSION
These preliminary data highlight the complexity of Nox function in lungs from CDH fetuses,
emphasizing that more than one Nox isoenzyme may be involved in Nox-mediated •O2
−
production.
Moreover, an altered H2O2 production regulated by Nox4 may suggest a malfunction of the
detoxification processes in the lungs. The raised oxidative stress seems to be a potential mechanism
involved on PH-associated with CDH.

28
MiRacles for babies with abnormal lungs: the story of miR-10a and lung development
R. Visser1, C. Fraser2, D. Mulhall2, F. Zhu2, C. Day2
, B. Iwasiow2, T. Mahood2,3, R. Keijzer1,2
1Department of General Surgery, University of Manitoba
2 Department of Pediatric Surgery and Child Health and Children’s Hospital Research Institute of
Manitoba, University of Manitoba; Winnipeg, MB, Canada
3 Department of Physiology and Pathophysiology, University of Manitoba; Winnipeg, MB, Canada
Corresponding author: richardkeijzer@gmail.com
Purpose
Worldwide, 150 babies are born every day with congenital diaphragmatic hernia (CDH). One
third of these infants will die from respiratory failure due to pulmonary hypoplasia.
MicroRNAs are essential epigenetic factors for lung development. We identified microRNA
miR-10a as a key regulator in CDH. We aimed to define the role of miR-10a in both normal and
abnormal lung development.
Methods
Using the nitrofen rat model to induce CDH, we employed real-time quantitative polymerase
chain reaction (RT-qPCR) and fluorescent in situ hybridization to study miR-10a expression
during development. Control- and nitrofen-treated fetal rat lungs were extracted for explant
cultures and treated with miR-10a inhibitors and mimics. We are investigating miR-10a’s
interaction with the retinoic acid signalling pathway using RT-qPCR and will confirm its
participation in vitro using a retinoic acid response element (RARE) dual luciferase assay.
Results
We found that miR-10a expression is localized in the mesenchyme of fetal lungs. Nitrofen
treatment reduces miR-10a’s expression, particularly early in gestation. Using nitrofen-treated
fetal rat lungs as explant cultures, we could reverse the hypoplastic phenotype by treating
lungs early in development with a miR-10a mimic. We are currently using RT-qPCR and the
RARE dual luciferase assay to confirm that miR-10a affects lung development through the
retinoic acid signalling pathway.
Conclusion
In CDH, reduced expression of miR-10a early in development contributes to pulmonary
hypoplasia. Treating developing lungs with miR-10a mimics can reverse the hypoplastic
phenotype. We believe this microRNA can help to develop a prenatal treatment to improve
the outcomes of CDH babies.

29
MiRacles for babies with abnormal lungs: the role of miR-200b in lung development
R. Visser1, N. Khoshgoo2, Vinaya Kumar Siragam2,3
, Barbara Iwasiow2,3
, Fuqin Zhu2,3
, Arzu Öztürk4,5
,
Sujata Basu3
, Molly Pind4,5
, Agnes Fresnosa4,5
, Gerald Stelmack3
, Geoff Hicks4,5
, Andrew Halayko3
,
Richard Keijzer1,2,3
1Department of General Surgery, University of Manitoba; Winnipeg, MB, Canada
2 Department of Pediatric Surgery and Child Health and Children’s Hospital Research Institute of
Manitoba, University of Manitoba; Winnipeg, MB, Canada 3Department of Physiology and
Pathophysiology, University of Manitoba; Winnipeg, MB, Canada
4
Manitoba Institute of Cell Biology University of Manitoba, Winnipeg, Canada,
5
Department of Biochemistry & Medical Genetics, University of Manitoba, Winnipeg, Canada
Corresponding author: richardkeijzer@gmail.com
Purpose
Respiratory failure is the leading cause of neonatal death in infants with congenital diaphragmatic
hernia (CDH). Reduced airway branching and a thickened layer of connective tissue characterize the
lungs of CDH babies. One explanation for the pathogenesis of CDH is defective epithelial-to-
mesenchymal interactions (EMT) during development. We have identified that miR200b is
abnormally expressed in CDH lungs. This microRNA is well known as an epigenetic moderator of EMT
in oncology. We aimed to define the effects of miR-200b on EMT during normal and abnormal lung
organogenesis.
Methods
We developed a miR-200b knockout mouse that allowed us to localized miR-200b expression with a
LacZ reporter gene. We then characterized both lung branching and pulmonary function in fetal and
newborn mice. By crossing our miR-200b knockout mice with CFP-ECadherin mice, we were able to
study the pulmonary epithelial phenotype in the presence and absence of miR-200b expression.
Results
LacZ staining revealed high miR-200b expression in fetal lungs. Heterozygous knockout lungs showed
reduced branching. Homozygous mice had decreased pulmonary function after birth. CFP-ECadherin
miR200b knockout lung explants indicated increased epithelial expression, consistent with altered
EMT signaling.
Conclusion
Epithelial-to-mesenchymal signaling is essential to proper lung development. Our findings support
that miR-200b is a key regulator of the crosstalk between the multiple pathways that participate in
this capacity. By altering the expression of miR-200b early in development, we believe this microRNA
could be the key to developing a prenatal treatment to improve the outcome in CDH babies.

30
DOWNREGULATION OF HEPATIC ADIPOSE DIFFERENTIATION-RELATED PROTEIN (ADRP) IN
NITROFEN INDUCED CONGENITAL DIAPHRAGMATIC HERNIA MODEL.
Takahashi H.1
, Friedmacher F.1
, Takahashi T.1
, Puri P.
1
National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland,
2
Conway Institue of Biomolecular and Biomedical Research, School of Medicine & Medical Science,
University College Dublin, Dublin, Ireland
Background: Vitamin A plays an essential role in biologic processes during embryogenesis.
Disruption of vitamin A homeostasis is a part of pathogenesis of congenital diaphragmatic hernia
accompanied by lung hypoplasia in mammalian and human cases. Since Vitamin A is not be
synthesized by the body, mammals absorb all Vitamin A from diet. After absorption, Vitamin A
transported to the lipid droplet in hepatic stellate cells for storage. Adipose differentiation-related
protein (ADRP) stabilize lipid droplet interface, which is a reliable and sensitive marker for lipid
droplets.
The teratogen induced nitrofen model has been widely used to investigate the CDH-associated
pathology in rodents. It has been reported that nitrofen disturbs the mobilization of retinoid from
placenta to fetus. However, retinoid storage in the hepatic lipid droplet in the CDH nitrofen model
remains unclear. We designed this study to test the hypothesis that the hepatic ADRP expression is
downregulated in the nitrofen-induced CDH model.
Methods: Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9).
Fetuses were harvested on D21, and livers were divided into two groups: controls and nitrofen (n =
24 at each time-point). ADRP gene expression level was analyzed by qRT-PCR.
Immunohistochemistry were performed to investigate ADRP protein expression.
Results: ADRP gene expression was significantly reduced in liver (ADRP: 1.72±0.19 vs. 0.99±0.10;
p=0.00721) and protein expression was markedly decreased in liver.
Conclusion: Our findings of downregulation of hepatic ADRP gene expression, suggest that the
retinoid storage in hepatic lipid droplets may be impaired in the nitrofen-induced CDH model.

31
Minimal invasive lung support via umbilical vein with a double-lumen cannula in a neonatal lamb
model: A proof of principle
F.Schmidt1
, J.Kuebler2
, M.Ganter3
, T.Jack1
, L.Meschenmoser4
, M.Sasse1
, M.Boehne1
, H.Bertram1
,
P.Beerbaum1
, H.Koeditz1
1 Department of Pediatric Cardiology and Intensive Care Medicine, Medical School Hannover, Carl-
Neuberg-Strasse 1, 30625, Hannover, Germany
2 Department of Pediatric Surgery, Medical School Hannover, Germany
3 University of Veterinary Medicine Hannover Foundation, Germany
4 Department of Cardiothoracic Surgery, Transplantation and Vascular Surgery, Medical School
Hannover, Germany
Corresponding author: Schmidt.florian@mh-hannover.de
Purpose: Acute respiratory distress syndrome, with the need for invasive mechanical ventilation
(MV), remains a major cause of neonatal mortality and morbidity. Although veno-venous
extracorporeal lung support (VV-ECLS) has become a standard of care procedure in neonatal
patients with acute pulmonary failure there are no reports regarding the use of a double-lumen
cannula for extracorporeal minimal invasive lung support via the umbilical vein.
Methods: A near-term neonatal lamb model was used (n=3). Umbilical vein was cannulated with a
double-lumen catheter allowing veno-venous extracorporeal gas exchange. Cannula was positioned
with its tip in the right atrium. VV-ECLS was started and ventilation was stopped. Providing
oxygenation and CO2 removal solely through VV-ECLS hemodynamics, blood gases were measured.
Results: Total VV-ECLS without MV was applied to all three neonatal lambs. Time on veno-venous
ECLS was 60, 120 and 120min. Initial pCO2 was 60, 55 and 65 mmHg compared to 31, 32 and 32
mmHg at the end of VV-ECLS. Initial pO2 was 30, 27 and 26 mmHg compared to 22, 19 and 23 mmHg.
Initial lactate was 5, 10 and 4 mmol/l compared to 13, 13, 11 mmol/l at the end of VV-ECLS. MAP at
baseline was 51, 55 and 65 mmHg compared to 36,38 and 41 mmHg at the end of VV-ECLS. In all
three lambs inotropes were admitted to maintain MAD >35mmHg.

32
Conclusion: VV-ECLS using a double lumen catheter via the umbilical vein can sufficiently remove
pCO2 without MV in a neonatal lamb model for a period of at least 120min. pO2 were measured 22,
19 and 23mmHg respectively at the end of VV-ECLS. At least partially caused by recirculation
phenomena, which could possibly be improved by different cannula design. Inotropic support was
necessary during VV-ECLS to achieve targeted MAD > 35mmHg.

33
Outcome of esophageal atresia/tracheoesophageal fistula in extremely-low-birth-weight neonates
(<1000 grams)
Augusto Zani, Giovanni Cobellis, Justyna Wolinska, Priscilla Chiu, Agostino Pierro
Division of General and Thoracic Surgery, The Hospital for Sick Children, Toronto, Ontario, Canada
PURPOSE: Recent advances in intensive care have resulted in a high survival of extremely low birth
weight (ELBW, <1000 g) neonates. The management of ELBW infants born with esophageal
atresia/tracheoesophageal fistula (EA/TEF) remains challenging and controversial. In this study, we
reviewed our experience with the surgical management of these neonates at our institution over a
fifteen year period.
METHODS: Ethical approval was obtained (REB1000046653). Medical records of ELBW infants with
EA/TEF treated at our institution between 2000 and 2014 were reviewed. Demographics, associated
anomalies, operative approach and postoperative complications were reported. Data are reported
as median (range).
RESULTS: Of the 268 infants with EA/TEF managed at our institution during the study period, 8 (3%,
5 females) infants had a birth weight of 930 grams (540-995) and a gestational age of 28 weeks (23-
32). Seven infants had type-C EA/TEF and 1 type B. Patient details and outcome are reported in the
Table.
CONCLUSION: This is the largest series of <1000 gram EA/TEF neonates. The incidence of ELBW
infants with EA/TEF is rare (3%). The association of ELBW and EA/TEF results in high morbidity and
mortality. In our series, the cause of death is mainly related to complications not associated with
EA/TEF repair. Fistula ligation followed by delayed anastomosis seems to achieve a better outcome
in ELBW infants.
Ass. anomalies Surgery (DOL= day of life) Complications Outcome
1 PDA DOL 1: Trans-pleural fistula
ligation and Stamm
gastrostomy
DOL 120: Delayed anastomosis
Anastomotic leak +
stricture (dilated x1)
PO feeds
FU of 5yrs
2 VSD, PDA, ARM,
annular pancreas ,
omphalo-
mesenteric duct
DOL 1: Extra-pleural fistula
ligation, small bowel resection,
stoma, Stamm gastrostomy
Gastric leak and
peritonitis, bilateral IVH
Death
3 Truncus arteriosus DOL 5: Extra-pleural fistula
ligation DOL 16: truncus
arteriosus repair
Intraop. liver
hemorrhage due to
peritoneal drain
insertion
Death
4 Butterfly vertebra,
tethered cord,
VSD, PDA, PFO,
fused kidney
DOL 2: Primary anastomosis +
sigmoidostomy loop
colostomy
DOL 125: PSARP
DOL 208: stoma closure
Occlusive saphenous
thrombus
PO feeds
FU of 3yrs
5 Fused vertebrae,
tethered cord,
PDA, PFO, ASD,
Talipes
DOL: 1 Trans-pleural fistula
division
DOL 5: Re-thoracotomy for
?air leak,
primary anastomosis
RDS, persistent
pneumothorax
multiple cerebellar and
brain hemorrhages -
palliated
Death
6 Trisomy 18 No surgery - palliated - Death
7 ASD, tethered cord DOL 1: : Extra-pleural fistula Thoracic duct injury,

34
ligation
DOL 60: Stamm gastrostomy
DOL 178: Delayed anastomosis
DOL 416: Lap Nissen
fundoplication
Pseudomonas
pneumonia, pulmonary
hypertension,
anastomotic strictures,
GERD
G-tube
feeds
8 PDA, RDS,
disseminated
fungemia
DOL 61: Stamm gastrostomy
DOL 126: TEF ligation and
delayed
anastomosis
DOL 217: Aortopexy (open)
Complications of severe
prematurity,
anastomotic strictures,
GERD
PO feeds
FU of 3yrs

35
TITLE: MODELLING THE INTERACTION OF COMORBIDITIES PREDICTING RISK OF FUNDOPLICATION
AUTHOR(S): Dr E.W. Macharia-Coates
AFFILIATION: Institute of Child Health, University College London, WC1N 1EH, United Kingdom
Email: eve.macharia-coates@ucl.ac.uk
PURPOSE
Prior studies of fundoplication risk focus on individual effects of comorbid risk factors. However, in
vivo, children with gastro-oesophageal reflux disease (GORD) may have multiple and interacting
pathologies, whose risk can be estimated. Our aim is to model fundoplication risk incorporating a
search for these interacting comorbidities.
METHODS
Data mining of an electronic clinical documents database was conducted. A retrospective 13-year
cohort of children (0-18 years) on prescription anti-reflux medications reviewed at a single
institution was identified. Putative risk factors identified i.e. demographics, comorbidities and
investigations. Exposure of interest was first fundoplication. Data partitioned into training and
testing datasets. Multivariable logistic regression with a search for 2-way interactions used to model
patient-specific fundoplication risk.
RESULTS
1080 patients (7.7%) of the cohort (n=13902) underwent
fundoplication between January 2000 – December 2012.
There were 29 associated comorbidities.
Table 1: Variables predicting risk of fundoplication. Beta-
coefficients for significant comorbidities (p<0.05) are re-
expressed as odds ratios (OR) and confidence intervals
(CI).
Statistical inference: In multivariate regression, 10
variables were significantly associated with an increase in
fundoplication risk. Additionally, we identified 6
interaction terms. Notably, neurological impairment (NI)
with prematurity decreases fundoplication risk.
Model performance: The model exhibited a sensitivity of
73% and specificity of 80%. The receiver operating
characteristic (ROC) analysis demonstrated an area under
the curve of 0.82. The model is a “good” predictor of
fundoplication risk.

36
Figure 1: ROC analysis for multivariate logistic regression
model incorporating 2-way interaction terms
Independent variables OR 2.5%CI 97.5%CI
ni 10.05 6.79 15.09
cdh 7.69 4.32 13.50
achalasia 7.18 2.28 19.88
renal 5.93 2.38 14.53
oatof 5.83 3.67 9.13
skeletal 5.66 2.44 13.43
prem 4.51 2.93 6.97
dental 3.76 0.99 13.31
cld 2.80 1.92 4.06
metabolic 1.81 1.04 3.07
Interaction terms
ni:cleft 108.51 9.53 4242.43
ni:swallow 12.80 3.00 91.39
ni:tracheal 10.79 3.16 45.72
cld:metabolic 7.38 1.15 47.16
ni:cardiac 3.36 1.99 5.69
ni:prem 0.40 0.24 0.68
CONCLUSION
We observe comorbidities with no individual effects that, in concert with NI, dramatically increase
fundoplication risk. Modelling to include a search for interactions reveals hidden risk factors. This
analysis demonstrates the importance of modelling with in vivo assumptions i.e. interacting
comorbidities.

37
Morphological changes and altered vascular measurements in the chick embryo model following
exposure to cadmium.
A Kaskova Gheorghescu, J. Thompson
School of Medicine and Medical Science, University College Dublin, Belfield, Dublin 4, Ireland
Abstract
Purpose: Chick embryos exposed to teratogenic doses of cadmium acetate (CdAc) at H-H stage 16-17
are known to develop abnormal body axes, with exaggerated lordosis in the lumbosacral region and
subsequent ventral body wall defect. It has been noted previously that cadmium causes abnormal
angiogenesis. The purpose of this study was to describe and quantify anti-angiogenic effect of Cd
utilizing the chick embryo model.
Methods: After 60 hours incubation, chicks were explanted according to Dugan’s method and
treated with 5, 25, 50, 75 and 100 µl of 50 µmol CdAc. Equimolar sodium acetate 50 µl was given to
controls. Embryos were returned to the incubator and re-examined at 4, 8, 24 and 48 hours later.
The dose at which Cd produces an anti-angiogenic effect was established. Morphological
abnormalities were observed. Vascular and embryo measurements (length of the sinus terminalis,
the size of the area vasculosa, vessel density and crown-rump length) were performed. Cadmium
uptake within the chick embryo was quantified. Quantitative real-time RT-PCR was used to
determine the relative expression of Ang-2 and VE-cadherin.
Results: 50 μL of 50 μM CdAc produced an anti-angiogenic effect. In Cd explants, extra-embryonic
vasculature was present in all treated explants, but a disturbed pattern was seen,
omphalomesenteric vessels appeared retarded with reduced branching. The sinus terminalis,
appeared tortuous and vascular development in the AV appeared suppressed generally. Complete
extra-embryonic avascularity wasn’t observed. The length of the sinus terminalis, the size of the area
vasculosa, vessel density, and CRL of the embryos were significantly reduced in the Cd group at all
time points when compared with controls. Treatment with 50 μL of 50 μM of CdAc resulted in a
mean embryo (n=14) Cd concentration of 4.1 ± 1.2 μM and a mean concentration of 7.6 ± 1.0 μM in
the extra-embryonic membranes (n=14) at 8 hours. The relative mRNA expression levels of Ang-2
were increased in the Cd group at 4 hours. VE-cadherin expression was significantly decreased in the
Cd group compared to controls at 1 hour and 4
Conclusion: Vascular endothelium is a target of Cd toxicity. Cadmium exposure impairs vascular
development, as evidenced by reduction in vascular and embryo measurements. Association
between abnormal vasculature and abnormal embryos was very strong. Results clearly indicate that
abnormal extra-embryonic vasculature is linearly associated with the growth retardation of the
embryo.

38
Table 1 Effects of cadmium dose on embryo survival and gross vascular abnormality after treatment
at 60 hours (Hamburger-Hamilton developmental stages 16-17).
Treatment (50 μL) N Dead at 60+24h Vascular abnormality 60+24h (%
of survivors)
5 μM CdAc 10 3 (30%) 0 (0%)
25 μM CdAc 10 4 (40%) 0 (0%)
50 μM CdAc 10 3 (30%) 4 (57.1%)*
75 μM CdAc 10 7 (70%) 3 (100%)
100 μM CdAc 10 7 (70%) 3 (100%)
NaAc 10 2 (20%) 0 (0%)
Table 2 The effects of Cd on vascular development in the chick area vasculosa.
TREATMENT N Dead Disturbed pattern
(% of survivors)
4 hours
Control (NaAc)
Cd (CdAc)
140
170
6 (4.2%)
16 (%)
0 (0%)
73 (47.4%)***
8 hours
Control (NaAc)
Cd (CdAc)
134
154
3 (2.2%)
14 (%)
0 (0%)
71 (50.7%)***
24 hours
Control (NaAc)
Cd (CdAc)
131
140
6 (4.5%)
27 (%)
0 (0%)
78 (77.9%)***
48 hours
Control (NaAc)
Cd (CdAc)
125
113
5 (4.0%)
26 (%)
0 (0%)
69 (79.3%)***

39
Table 3 Gross abnormalities in embryos 48 hours after treatment at 60 hours.
ABNORMALITY
N
Limb abnormalities, including hypoplasia and malposition 7 (28%)
Longitudinal body axis malformation with dorsal kink in the caudal
end of the embryo
7 (28%)
Eye abnormalities, including microphthalmia and unilateral
anopthalmia
0 (0%)
Defects of the cranial part of the neural tube 0 (0%)
Facial abnormalities, including midfacial cleft and hypoplasia 0 (0%)
Figure 1 The length of the sinus terminalis.

40
Figure 2 The size of the area vasculosa.
Figure 3 Vessel density.
Figure 4 Crown-rump length in control and Cd-treated embryos.

41
Figure 5 Relative mRNA expression levels of Ang-2 at 60 hours.
Figure 6 Relative mRNA expression levels of VE-cadherin at 60 hours.

42
SCIENTIFIC SESSION III

43
Non-Invasive Ablation of Fetal Rabbit Tissue In Utero Using Magnetic Resonance Guided High
Intensity Focused Ultrasound
K. Piorkowska1
, A.C. Waspe1
, C. Mougenot2
, T. Wang3
, J.T. Gerstle1,4
and J. Drake,1,5
.
1
Centre for Image Guided Innovation and Therapeutic Intervention, Hospital for Sick Children,
Toronto, Ontario, Canada.
2
Philips Healthcare, Toronto, Ontario, Canada.
3
Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario,
Canada.
4
Department of General and Thoracic Surgery, Hospital for Sick Children, Toronto, Ontario, Canada.
5
Department of Neurosurgery, Hospital for Sick Children, Toronto, Ontario, Canada.
Email of Corresponding author: Karolina.Piorkowska@sickkids.ca
PURPOSE: Magnetic resonance guided high intensity focused ultrasound (MRgHIFU) is a potential
non-invasive therapy for fetal conditions including lung malformations and other solid organ lesions
requiring invasive surgical interventions. This acute study assessed feasibility and safety parameters
of in utero MRgHIFU delivery and determined accuracy and tissue response to treatment.
METHODS: High resolution 3T MR images were acquired in late gestation rabbits (~30 days, term =
32; n=5) to identify clinically relevant fetal targets. HIFU sonications were applied continuously for
~20 seconds to a specific target organ at various powers to achieve areas of necrosis guided by MR
and thermal imaging. HIFU exposures are presented as average acoustic power in Watts (Wac) ±
standard deviation reaching maximum temperatures, averaged over all exposures, in degrees
Celsius. Tissues were collected for histological analysis.
RESULTS: Lungs (n=8) were targeted with 85 ± 15 Wac reaching 74°C and livers (n=6) with 80 ± 15
Wac reaching 75°C. Histological changes for both tissue types showed distinct necrotic areas with
circumferential hemorrhage which transitioned to healthy tissue. In one fetus, a “banding pattern”
occurred due to rib interference. Kidneys (n=5) were treated with 100 Wac and reached 67°C: one
successful treatment resulted in a focal area of necrosis and surrounding vasodilation of arteries;
other treatments were less effective due to fetal movement and high perfusion of the kidney.
Necropsy revealed mild maternal skin burns (n=2) that was rectified by improved coupling and
increased skin cooling time; also, mild architectural changes in the smooth muscle of the external
uterine wall (n=3) not spanning the breadth of tissue layers to the internal surface. Examination of
the fetuses revealed focal epidermal vasodilation on the chest wall of fetus (n=8) associated with
lung targeting. Collateral organ damage ranged from mild vasodilation to diminutive necrosis and
was due to fetal size and overlapping orientation of organs. There was no apparent affect to
adjacent untreated fetuses.
CONCLUSION: MRgHIFU therapy can effectively target and thermally treat specific in utero organs in
this acute fetal rabbit model. Clinical MRgHIFU therapy for specific organ anomalies may improve
overall fetal outcome over traditional invasive surgical procedures.

44
HUMAN ADIPOSE MESENCHYMAL CELLS SUPPRESS MELANOCYTE FUNCTION AND SKIN
PIGMENTATION BY SECRETING TRANSFORMING GROWTH FACTOR β-1
A. S. Klar1,2
*, T. Biedermann1,2
*, K. Michalak1,2
, T. Michalczyk1,2
, C. Meuli-Simmen3
, M. Meuli2,4
, E.
Reichmann1,2
(1) Tissue Biology Research Unit, Department of Surgery, University Children’s Hospital Zurich,
Zurich, Switzerland
(2) Children’s Research Center, University Children’s Hospital Zurich, Zurich, Switzerland
(3) Department of Plastic, Reconstructive, Esthetical and Hand Surgery, Kantonsspital Aarau, Aarau,
Switzerland
(4) Department of Surgery, University Children’s Hospital Zurich, Zurich, Switzerland
*authors contributed equally
Correspondence should be addressed to: Ernst.Reichmann@kispi.uzh.ch
Purpose: We have previously shown that skin pigmentation is determined by mesenchymal-
epithelial interactions via the interplay of transcription factors and growth factors influencing skin
melanocytes. In this experimental study, we investigated the effect of adipose-derived stromal cells
(ASCs) on the melanocyte functions such as differentiation, proliferation, melanogenesis, and
dendritogenesis in dermo-epidemal skin substitutes.
Methods: Human epidermal melanocytes, keratinocytes, and fibroblasts were isolated from dark-
pigmented skin biopsies, whereas ASCs were isolated from human adipose tissue biopsies. After in
vitro cell expansion, bovine collagen hydrogels containing ASCs or fibroblasts were prepared, and
melanocytes and keratinocytes were seeded in a 1:5 ratio onto those hydrogels. The dermo-
epidermal skin substitutes were transplanted onto full-thickness wounds of immuno-deficient rats
and analyzed after 5 weeks.
Results: ASCs significantly suppressed the growth and pigmentation of melanocytes in dermo-
epidermal skin substitutes as compared with skin-fibroblasts. ELISA and western blot analyses
showed higher levels of TGFβ-1 in ASCs as compared to skin-fibroblasts. We showed that
melanocytes respond to TGFβ-1 in vitro and in vivo by suppressing cell proliferation and the
expression of melanosomal markers, such as tyrosinase and tyrosinase-related protein 1, which are
essential for maturation of melanosomes and melanin synthesis. Furthermore, the distribution of
melanin in the epidermis was also suppressed as demonstrated by Fontana Masson staining.
Conclusions: Our data suggest that mesenchymal cells of different tissue origin - adipose and skin -
used to construct pigmented dermo-epidermal skin substitutes, have distinct influences on the
melanocyte functions and skin pigmentation. Adipose-ASCs significantly decreased melanocyte
function that resulted in generation of light-pigmented skin substitutes, which did not correspond to
the dark-pigmented donor skin color.
Keywords: Melanocytes - Melanogenesis - Adipose-derived stromal cells - TGFβ-1 - Skin tissue
engineering - Pigmented skin substitutes - Rat model

45
Colonic epithelia disruption after maternal separation is rescued by corticotropin releasing
hormone
Bo Li1
, Tali Filler1
, Carol Lee1
, Augusto Zani1
, Elke Zani-Ruttenstock1
, Wan Ip2
, Tanja Gonska2
, Agostino
Pierro1
1
Division of General and Thoracic Surgery, The Hospital for Sick Children, Toronto, ON, Canada
2
Department of Pediatrics and Gastroenterology, The Hospital for Sick Children, Toronto, ON,
Canada
PURPOSE: Early maternal separation (MS) induced colonic disruption of morphology and
permeability, enhancing the risk of early intestinal disorders. This effect could be associated with
brain-gut interactions through the release of corticotropin releasing hormone (CRH) by
hypothalamus. The aim of the present study was to determine whether intestinal epithelial damage
could be restored by modulation of the CRH pathway.
METHODS: Following approval (license 32238), C57BL/6 neonatal mice underwent MS for 3 hours
daily between postnatal day 5 and 9. Mice were randomly assigned to receive an intraperitoneal
injection of: i) DMSO (MS+DMSO group, n=10), or ii) Antalarmin, a CRF antagonist (10
mg/kg/day, MS+Antalarmin group, n=10). MS was performed after treatment with DMSO
or Antalarmin. Untreated pups served as control. Proximal colon (most injured gut area) was
harvested and analyzed for goblet cell density per crypt (alcian blue), crypt length (H&E) and tissue
permeability. Ussing chamber was used to test tissue viability (trans-epithelial resistance) and trans-
cellular flux. Data was compared using one-way ANOVA with Bonferroni post-test; p<0.05 was
considered significant.
RESULTS: Morphology - Compared to controls, the number of goblet cells per crypt was reduced in
MS+DMSO mice (p<0.001), but increased by Antalarmin (p<0.01 to MS+DMSO; Figure 1a). Similarly,
colonic crypt length was decreased in MS+DMSO mice (p<0.01 to control), but returned to normal
levels after Antalarmin administration (p<0.05; Figure 1b). Permeability - Trans-epithelial resistance
remained unchanged across the groups confirming tissue viability of all samples. MS+DMSO
increased trans-cellular permeability compared to control (p<0.001) but MS+Antalarmin reduced it
to level observed in control (p<0.001) (Figure 1c).
CONCLUSION: Colonic mucosal damage and increased trans-cellular permeability induced by
maternal separation are rescued by Antalarmin, a CRH antagonist. These findings suggest that
maternal separation induced bowel damage could be due to a disruption of the gut-brain axis. This
provides insights on the development of new drugs that could be used for the treatments of
neonatal intestinal diseases, such as necrotizing enterocolitis.

46
AUTOPHAGY AS A CHARACTERISTIC FEATURE OF NEONATAL TISSUE MACROPHAGES
Authors: T. Winterberg1
, Y. Yu1
, S. Groos2
, J.K. Park1
, G. Vieten1
, B. Ure1
, JF. Kuebler1
Institutional affiliations
1
Department of Pediatric Surgery
2
Institute of Cell Biology in the Center of Anatomy
Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover
E-mail address
Kuebler.Joachim@mh-hannover.de
Purpose: Macrophage autophagy has been the focus of recent interest and a growing body of
evidence indicates that it is a key component of the innate immune response, involved in many
aspects of macrophage development and polarization. In former studies we observed distinct
phenotypic and functional features of neonatal tissue macrophages. The aim of this study was to
investigate the occurrence of autophagosomes in neonatal macrophages.
Methods: Peritoneal macrophages were gained by injury-free lavages of neonatal and adult C57/BL6
mice. Pelleted cells were investigated by electron microscopy. FACSorted, purified macrophages
were stained using standard May-Grünwald-Giemsa staining or further cultured and stimulated for
6h with TLR agonists. Total mRNA was extracted and detected using Agilent Micro Array Detection
System. After data normalization specific software was used to investigate primarily homeostatic,
pro- and anti inflammatory processes.
Results: Naive neonatal macrophages displayed a high amount of cytoplasmic vesicular structures
visible after staining (Fig.1). Electron microscopy of macrophages from neonatal mice regularly
showed autophagosomes (Fig. 2B). Microarray gene network analysis revealed that the increase in
autophagy was associated with a signature of increased homeostatic activity as well as a decrease in
genes responsible for resolving inflammation (Alox15, Tgfb2, Ikbkg).
Conclusion: Neonatal macrophages appear to display an increased autophagic activity in
combination with a tissue and developmental homeostatic “priming”. Although this seems to be
well adapted to the major changes in the tissue microenvironment after birth, these changes could
have adverse effects on the immunological balance by down regulation of pro-resolving and anti-
inflammatory mediators.
Fig.1 Fig.2

47
Fig.1 Naive neonatal (A) and adult (B) peritoneal macrophages (May-Grünwald-Giemsa staining; one
representative experiment of >8 experiments;
bars: 10 µm.
Fig.2 Thin sections of naive neonatal (A, B) and adult (C, D) peritoneal macrophages; (AP)
autophagosome, asterisks: Endoplasmic Reticulum, (G) Golgi stack, (M) Mitochondrium, (N) nucleus;
bars A, C: 2 µm; B, D: 1 µm.

48
Modified epidermal self renewal of engineered pigmented human skin after UVB exposure in an in
vivo assay
Teresa Michalczyk, Thomas Biedermann, Sophie Böttcher- Haberzeth,
Agnes Klar, Ernst Reichmann, Martin Meuli
T. Michalczyk, T. Biedermann, S. Böttcher- Haberzeth, A. Klar, E. Reichmann
Tissue Biology Research Unit, University Children’s Hospital Zurich, Zurich, Switzerland
S. Böttcher- Haberzeth, M. Meuli
Departement of Surgery, University Children’s Hospital Zurich, Zurich, Switzerland
T. Michalczyk, T. Biedermann, S. Böttcher- Haberzeth, A. Klar, E. Reichmann, M.Meuli
Children’s Research Center, University Children’s Hospital Zurich, Zurich, Switzerland
e-mail: Teresa.Michalczyk@kispi.uzh.ch
Abstract
Purpose The basal compartment of the human epidermis is constantly renewing, but still little is
known about the epidermal mechanism of maintaining and adapting its homeostasis, in particular
when exposed to external stress factors like UV irradiation. UVB light provokes tanning in human
skin. Also, it can lead to inflammation and can induce mutagenic events in the genome of skin cells.
We recently demonstrated successful engineering of pigmented dermo-epidermal skin substitutes,
with restoration of the original donor skin color in long-term in vivo experiments. In view of future
clinical application, we now studied the behavior of these tissue-engineered substitutes after
exposure to UVB irradiation.
Methods Human dermo-epidermal pigmented skin was engineered with keratinocytes, melanocytes
and fibroblasts originating of the same donor biopsy. Then, keratinocytes and melanocytes were
seeded on collagen type I hydrogels, previously populated with fibroblasts. Skin substitutes were
transplanted onto full-thickness wounds of immuno-incompetent rats and irradiated with a single
dose of 250mJ/cm2
UVB irradiation 4 weeks after transplantation. Punch biopsies were taken from
the grafts before and after irradiation for immunohistochemical staining.
Results After UVB irradiation transplanted skin substitutes showed physiological tanning properties.
Temporarily, Cytokeratin 16 and Desmoglein 3 were expressed in keratinocytes, indicating a wound
healing response. In engineered skin without UVB exposure, as well as in foreskin, few proliferating
keratinocytes were mostly located in the suprabasal layer, directly adjacent to the basal layer. After
UVB exposure proliferation of keratinocytes, but not melanocytes or fibroblasts, massively increased
notably in the basal layer and normalized 4 weeks after irradiation. The expression of the Wnt-
pathway inhibitors Dickkopf 3 and Wif 1 was lost in basal keratinocytes after UVB irradiation,
authorizing proliferation of keratinocytes. Interestingly, melanocytes continued to express Wnt-
Inhibitors after UVB treatment.

49
Conclusion These findings suggest that our engineered human skin is capable to maintain epidermal
homeostasis after external stress, via modified controlled proliferation of keratinocytes. This is
crucial regarding a future clinical application of tissue-engineered pigmented autologous skin
substitutes on patients. Furthermore, there is evidence that the basal compartment itself is
balancing inhibitors of the Wnt-pathway to initiate epidermal self-renewal.

50
Comparison of different methods of preservation to store decellularised oesophagi for tissue
engineering purposes
L. Urbani1
, P. Maghsoudlou1
, A. Milan1,2
, S. Eaton1
, P. De Coppi1
.
1
UCL Institute of Child Health and Great Ormond Street Hospital, UCL, London, UK
2
University of Padua, Padua, Italy
Corresponding author: l.urbani@ucl.ac.uk
Purpose
Several conditions may require esophageal tissue replacement. Tissue engineering of the
oesophagus (TEO) has been proposed as a therapeutic alternative to oesophageal substitution.
Acellular matrices are ideal for TEO because they are made up by tissue-specific extracellular matrix
(ECM). However, the appropriate preservation of the ECM components may significantly affect the
behavior of scaffolds in vivo. Thus far, there is no consensus on the most appropriate storage
methodology for decellularised scaffolds. Here we aim at establishing the best way to storage
oesophageal scaffolds from a large animal model and preserve the structure of their ECM.
Methods
Rabbit oesophagi were decellularized using detergent-enzymatic treatment (DET) and evaluated at 2
weeks, 1, 3 and 6 months of storage by means of histology, immunofluorescence, biomechanical
testing, ECM component quantification and angiogenic assay. Four different storage methods were
compared: A) phosphate-buffered saline solution at 4°C, B) at -20°C after a freeze-drying step, C) at -
80°C after snap-freezing in liquid nitrogen or D) in liquid nitrogen after cooling in medium with 10%
DMSO at -1°C/min then stored in liquid nitrogen.
Results
Efficient decellularization was achieved after 2 cycles of DET as evidenced by histology and DNA
quantification, with preservation of collagen, elastin and glycosaminoglycans levels. As a storage
methodology the PBS/4°C option (method A) led to the most structural destruction of the tissue,
whereas gradual freezing of the tissue (method D) was best at preserving the tissue for long term.
Scaffolds stored with method D had a preserved structure and orientation throughout all the time
points, showing intact layers and collagen and elastin fibers. Oesophagi stored at -80°C post snap-
freezing (method C) were substantially different compared to the rest, displaying irreversible
collapse and breaking up of the tissue.
Conclusion
Developing of an acellular scaffold for oesophageal tissue engineering that preserves the ECM
components and architecture of the original tissue is essential for therapy. Efficient storage allows
their use as an ‘off-the-shelf’ product. This study demonstrated that, similarly to cellular
cryopreservation, slow cooling in a DMSO/medium solution and subsequent storage in liquid
nitrogen allows long term storage of decellularized scaffolds.

51
Development of an Extracellular Matrix Hydrogel for use in Intestinal Tissue Engineering.
C.Crowley1
, L.Urbani1
, V.Gaillet1
, F.Onofri1
, L.Maughan1
, S.Eaton1
,P.Coppi1
.
1
UCL Institute of Child Health and Great Ormond Street Hospital, UCL, London, UK
Corresponding author: claire.crowley.09@ucl.ac.uk
Abstract:
Purpose: Irreversible intestinal failure (IF) is a condition that can lead to significant morbidity and
mortality and occurs as a result of anatomical or functional loss. Perenteral nutrition has been
shown to improve the condition substantially, however, there are many associated complications
with this approach. As a result, many patients eventually require an intestinal transplant, which is
unfortunately limited by the availability of donor organs and the need for aggressive
immunosuppression. Recent years have brought many advances to this field particularly regarding
isolation and culture of intestinal stem cells. However, their expansion for translational application
remains limited, so the discovery of alternative culturing methods is crucial. Here we investigate the
ability to create a hydrogel from the extracellular matrix (ECM) of intestinal tissue. ECM hydrogels
from other tissues have shown great potential for many different applications. This study aims to
develop and characterise an intestinal ECM hydrogel for eventual use in intestinal organoid culture
and as a hybrid scaffold for tissue engineered intestinal replacement.
Methods: A decellularisation protocol was developed specifically for newborn porcine intestinal
tissue. This method was assessed and characterised using SEM, histology and quantification of
collagen, elastin, GAG and DNA content after each cycle. The decellularised tissue was then
lyophilised, milled into a powder, pepsin digested and brought to a physiological pH and
temperature to produce a soluble ECM gel. The gel was characterised for its rheological properties
and gelation potential using spectrometry and oscillatory tests. Whole intestine containing both the
muscle and mucosa and the mucosa alone were compared, as this method can be highly tissue
specific.
Results:
One cycle of the decellularisation protocol was sufficient to successfully remove the nuclei and
reduce the DNA content of the intestinal tissue. Collagen, GAG and Elastin remained intact after
decellularisation. An ECM hydrogel was successfully produced and characterised. Optimal conditions
(pH, concentration, timing) for gel formation were established to obtain a hydrogel suitable for cell
culture and in vivo application.
Conclusion: In this study we established a tissue specific ECM-derived hydrogel for intestinal tissue
engineering. The applications for these gels is extensive, both for clinical and research purposes.

52
Quantitation of residual detergent in decellularised organs for tissue engineering with gas
chromatography-mass spectrometry
E. Maughan1
, C. Crowley1
, R. Bhondi1
, F. Tommasini1
, L. Urbani1
, C. Butler1
, M. Birchall1
, S. Eaton1
, P.
De Coppi1
.
1
Institute of Child Health and Great Ormond Street Hospital, UCL, London, UK
Corresponding author: claire.crowley.09@ucl.ac.uk
Purpose
Sodium deoxycholate (SDC) is a cytotoxic detergent widely used in organ decellularisation for tissue
engineering for the removal of cell membrane components. It has been reported that residual SDC
can compromise cell viability during subsequent recellularisation, 3D culture and implantation.
However, quantification of residual SDC in the tissue has not been optimized. This study was
designed to evaluate the use of Gas Chromatography-Mass Spectrometry (GC-MS) in determining
the efficacy of residual SDC removal following post-decellularisation washing.
Methods
Rat tracheae (n=16) were decellularised using a detergent-enzyme technique containing SDC over
three cycles. Following decellularisation, a washing step using double-distilled water (MilliQ) was
performed with agitation for 24 hours, 48 or 72 hours. Wash solutions were changed every 24 hours.
Samples were acidified, cholic acid added as an internal standard, extracted with diethyl ether,
derivatised to the pentaflurobenzyl, trimethylsilyl derivative, and analyzed by GC-MS.
Results
GC-MS proved a highly sensitive method for detection of very small residual concentrations of SDC
(as low as 2nmol). The concentration of residual SDC was cumulative with increasing cycle number
(1.64 vs 3.91 vs 9.56 nmol for cycles 1, 2 and 3 respectively). Increasing washing times resulted in a
demonstrable decrease in SDC concentration (e.g. 9.56 to 3.56 nmol from 24- to 72-hour
timepoints).
Conclusion
The novel use of GC-MS as a highly sensitive method for detecting and quantifying residual SDC
within decellularised tissue could enable the comparison of washing protocol efficacy. Further
validation of this analytical tool could enable its development as a GMP release criteria for
decellularised tissue in clinical trials.

53
CYTOREDUCTIVE SURGERY (CRS) AND HYPERTHERMIC INTRAPERITONEAL CHEMOTHERAPY (HIPEC)
IN PEDIATRIC OVARIAN TUMORS: A NOVEL TREATMENT APPROACH
A Hayes-Jordan 1
, CLopez2
, HL Green1
, LC Xiao3
, W Huh MD4
, C Herzog 4
ahjordan@mdanderson.org
1. University of Texas MD Anderson Cancer Center, Department of Surgical Oncology/Pediatric
Surgical Oncology, Houston, Texas, USA 2. University of Texas Houston Health Sciences Center 3.
University of Texas MD Anderson Cancer Center, Department of Biostatistics 4. University of Texas
MD Anderson Cancer Center, Division of Pediatrics
Purpose: CRS and HIPEC have been used in adults with ovarian carcinoma proving overall survival
benefit in randomized trials, measured in months. Diffuse peritoneal disease from pediatric type
ovarian tumors is rare. We applied this approach to a select group of pediatric girls with diffuse
peritoneal disease. These patients were all included as part of a phase 1 or phase 2 clinical trial for
CRS and HIPEC in children.
Methods: In all patients complete cytoreduction followed by HIPEC using 100mg/M2 of Cisplatin for
90 minutes in a closed technique, was utilized. All patients were treated with the same strict peri--
operative management, as part of an investigator initiated clinical trial. All received neoadjuvant
chemotherapy. Patients with disease outside of the abdominal cavity were excluded.
Results: Of 101 pediatric CRS and HIPEC operations, 8 had ovarian primary tumors and multifocal
peritoneal disease. There were 3 yolk sac tumors( germ cell, mixed teratoma), one Sertoli-Leydig,
one PNET of the ovary, one choriocarcinoma, one juvenile granulosa cell tumor and one
adenocarcinoma. Age at diagnosis ranged from 4 to 18 years. Two of the 7 (28%) recurred and died.
The remaining 70% are disease free 2 to 8 years post HIPEC. Overall survival and relapse free
survival in this cohort was 64% and 62% respectively. [CI 0.64 (0.34,1 ); 0.62 ( 0.37, 1 )]
Complications included 2 wound infections, and 1 urinary tract infection and 1 enterocutaneous
fistula.
Conclusions: This is the first report of CRS and HIPEC in pediatric ovarian tumors. HIPEC is a safe
approach to diffuse peritoneal disease secondary to pediatric-type ovarian tumors. More treated
patients are required to determine efficacy of this approach.

54
SCIENTIFIC SESSION IV

55
PLATELET-LEUKOCYTE FIBRIN MEMBRANES AS POTENTIAL PLATFORMS FOR WOUND HEALING
*
F. Grandi1
, E. Stocco2,5
, S. Barbon2,5
, S. Capelli3
, A. Borean3
, A. Porzionato4
, V. Macchi4
, G. Albertin4
,
R. De Caro4
, PG. Gamba1
, P.P. Parnigotto5
, C. Grandi2
1
Pediatric Surgery, Department of Woman and Child Health, University of Padua, Padua, Italy
2
Department of Pharmaceutical an Pharmacological Sciences, University of Padua, Padua, Italy
3
Department of Immunohematology and Transfusion-Medicine of Belluno Hospital, Belluno, Italy
4
Section of Human Anatomy, Department of Molecular Medicine, University of Padua, Padua, Italy
5
Foundation for Biology and Regenerative Medicine, Tissue Engineering and Signaling (TES) ONLUS,
Caselle di Selvazzano Dentro, Padua, Italy
*
Corresponding and presenting author: Dr. F. Grandi francesca.grandi@studenti.unipd.it
PURPOSE Translational medicine has emerged as a new trend in medical practice; one of the main
topics deals with those therapies focused on enhancing tissue repair and regeneration. In particular,
the use of autologous haemocomponents as biomolecules delivering systems is gaining wide
attention. Recently, we developed a new method to collect haemocomponents for regenerative use,
obtaining gels rich in platelets, monocytes/macrophages, fibrinogen and CD34+
cells. Even though
these preparations are available today to stimulate tissue healing, they do not fully satisfy surgeons’
requests, looking for easily applicable and manipulable membranes. Hence, our aim was to
manufacture a novel Platelet-Leukocyte Fibrin membrane (PLFm) and to study its morpho-
mechanical properties as well as its attitude to act as a scaffold.
METHODS PLFms were prepared from a Platelet-Leukocyte concentrate/Plasma MIX as we
previously described(Caloprisco et al., Transfus Apher Sci. 2010;42:117-24) after activation with
calcium gluconate. The resulting PLFms were characterized haemocytometrically for cell
concentration in comparison with the MIX. To predict the in vivo behaviour of PLFms, supports were
incubated in PBS at 37 °C up to day 21, and their morphology/histoarchitecture were investigated by
histology, immunohistochemistry and SEM, at different end-points. In addition, contingent variations
in mechanical properties of PLFms were also assessed by tensile tests. Lastly, PLFms were used as
scaffolds to verify their ability in sustaining cell adhesion/proliferation.
RESULTS From a MIX volume of 11±2 ml were obtained membranes of 2.8±0.6 ml in volume with an
area of 8.9±1.4 cm2
and 0.31±0.06 cm in thickness. A significant increase in cell concentration was
observed for PLFms compared to the MIX. Histology and SEM showed that samples morphology
changed along with time, as the fibrin scaffold, previously masked by erythrocytes, became
progressively visible. The presence of cellular elements of interest was also assessed by
immunohystochemistry. Moreover, any significant difference in the maximum percentage of
deformation was observed within 10 days of incubation. Cell culture on PLFms showed the attitude
of these haemocomponents in sustaining cell adhesion/proliferation.
CONCLUSIONS Autologous PLFms represent a platform with structural and biological properties that
promote tissue healing and with a possible routinary application in surgery.

56
SPHINGOSINE-1-PHOSPHATE CONTROLS CELL MOTILITY OF PLACENTA MESENCHYMAL STEM CELLS
Giulio Innamorati#*
, Emanuela Fontana, Federica Steccanella, Giovanni Ridolfi, Kushal Gandhi, Luca
Giacomello*
Paediatric Surgical Research Laboratories, Department of Surgery, University of Verona, Italy
*Corresponding authors: giulio.innamorati@univr.it, luca.giacomello@univr.it
PURPOSE
A major concerns in using stem cells for therapeutic approaches is the very limited percent of
engraftment and, as a consequence, the large number of cells to be administered. Sphingosine 1
phosphate (S1P) is emerging as a crucial regulator of cell motility and chemotaxis acting in concert
with CXCL12 to regulate the egression immature progenitors. We explored the functional
implications of S1P signaling in placenta derived mesenchymal stem cells (PDMSC), a promising
opportunity for a number of diseases.
METHODS
Primary cultures of fetal cells were obtained from chorion of human term placenta. Mesenchymal
properties were confirmed by osteocytic and adypocytic differentiation. S1P receptors (S1PRs) gene
expression was assessed by RT-PCR utilizing specific primers for each of the 5 existing subtypes.
ERK1/2 and PKD1 activation was measured by western blot. Cell motility was monitored in DMEM
supplemented with 0.3% fetal calf serum, migration was digitally quantified after fixation and crystal
violet staining.
RESULTS
RT-PCR revealed mRNA expression of S1P receptors subtypes 1, 3, 4. Subtypes 2 and 5 were absent.
Consistently, SEW2871 and CYM50179 (selective agonists for S1P1R and S1P4R respectively) and
FTY-P (active on S1PR 1,3,5,4) induced ERK1/2 activation (A).
0%#
20%#
40%#
60%#
80%#
100%#
120%#
0# 60# 120# 180# 240# 300# 360# 420# 480# 540# 600# 660# 720#
0%#
20%#
40%#
60%#
80%#
100%#
120%#
SEW2871# CYM# FTY720# S1P#
C
YM
50179
S1P
**
**
**
FTY720PSEW2871
A
ERK1/2activation(%S1Pstimulated-basal)
FTY720P
SEW
2871
S1P
CYM 50179
B
ERK1/2activation
(%maximalstimulation-basal)
0%#
10%#
20%#
30%#
40%#
50%#
60%#
70%#
1.E-08# 1.E-07# 1.E-06# 1.E-05#
Woundclosure(%)
C
[S1P] (log M) -7 -6 -5Time (hours) 1 2 3 4 5 18 24

57
Addition of S1P promoted the transient activation of ERK1/2 (B). An analogous effect was observed
with PKD1. Wound healing assays revealed that increasing concentrations of S1P significantly and
progressively reduced PDMSC motility (C).
CONCLUSION
We demonstrated that PDMSC express more than one G protein coupled receptor specific for S1P.
S1P signaling inhibits cell migration and could be exploited to design novel approaches aimed to
promote PDMSC engraftment in the target tissues.

58
Effects of curcumin on a pediatric hepatocellular carcinoma model in vivo and curcuminoid
concentrations after oral application in mice
V. Ellerkamp1
, N. Bortl1
, J. Frank2
, C. Schiborr2
, S. Armeanu-Ebinger1
, E. Schmid1
, B. Kirchner1
, S.W.
Warmann1
, J. Fuchs1
University Children`s Hospital Tuebingen, Department for Pediatric Surgery and pediatric Urology,
Tuebingen, Germany
University Hohenheim, Institute of Biological Chemistry and Nutrition, Hohenheim, Germany
Abstract
In children with hepatocellular carcinoma (pHCC) the 5-year overall survival rate is poor. In adult HCC
several antitumor properties are described in in vitro models for the use of curcumin.
Methods: Orthotopic growth of the pediatric hepatocellular carcinoma cell line HC-AWF1 in
NOD/LtSz-scid/IL-2Rgamma(null)mice was induced. By the increase serum alpha fetoprotein AFP >5
U/mL mice were randomly assigned to one of four groups: control (no treatment); micellar
curcumin; cisplatin, and micellar curcumin + cisplatin. Curcuminoid levels in serum and organ lysates
as well as AFP serum levels were investigated.
Results: Serum curcumin decreased from 3513.89 ± 2791.84 nmol/L two hours after administration
to 769.74 ± 448.61 nmol/L after five hours. Curcumin concentrations significantly differed between
organs (p=0.000), highest concentrations were observed in the lungs 11.33 ± 9.17 nmol/Kg, lowest in
the brain 0.16 ± 0.24 nmol/Kg. The concentrations in the tumor tissue (2.57 ± 1.49 nmol/Kg) were
higher than in the liver (1.77 ± 1.50 nmol/Kg).
Combination therapy (micellar curcumin + cisplatin) significantly reduced AFP concentrations
compared to control group (week 3: 1.04 ± 0.67 vs. 2.73 ± 0.64, p = 0.004; week 4: 2.05 ± 1.01 vs.
3.35 ± 0.43, respectively, p = 0.02).
Conclusion: These data prove the potential of micellar curcumin as a complementary agent in
pediatric oncology to enhance the overall survival of patients with pediatric liver tumors.

-28th ISPSR POSTER

Empfohlen

Empfohlen

Weitere ähnliche Inhalte

Was ist angesagt?

Was ist angesagt? (20)

Andere mochten auch

Andere mochten auch (20)

Ähnlich wie -28th ISPSR POSTER

Ähnlich wie -28th ISPSR POSTER (20)

-28th ISPSR POSTER